The spliceosome-associated protein CWC15 promotes miRNA biogenesis in Arabidopsis

MicroRNAs (miRNAs) play a key role in regulating gene expression and their biogenesis is precisely controlled through modulating the activity of microprocessor. Here, we report that CWC15, a spliceosome-associated protein, acts as a positive regulator of miRNA biogenesis. CWC15 binds the promoters of genes encoding miRNAs (MIRs), promotes their activity, and increases the occupancy of DNA-dependent RNA polymerases at MIR promoters, suggesting that CWC15 positively regulates the transcription of primary miRNA transcripts (pri-miRNAs). In addition, CWC15 interacts with Serrate (SE) and HYL1, two key components of microprocessor, and is required for efficient pri-miRNA processing and the HYL1-pri-miRNA interaction. Moreover, CWC15 interacts with the 20 S proteasome and PRP4KA, facilitating SE phosphorylation by PRP4KA, and subsequent non-functional SE degradation by the 20 S proteasome. These data reveal that CWC15 ensures optimal miRNA biogenesis by maintaining proper SE levels and by modulating pri-miRNA levels. Taken together, this study uncovers the role of a conserved splicing-related protein in miRNA biogenesis.

The spliceosome-associated protein CWC15 promotes miRNA biogenesis in Arabidopsis Bangjun Zhou 1,2 , Huihui Yu 1,2 , Yong Xue 1,2 , Mu Li 1,2 , Chi Zhang 1,2 & Bin Yu 1,2 MicroRNAs (miRNAs) play a key role in regulating gene expression and their biogenesis is precisely controlled through modulating the activity of microprocessor.Here, we report that CWC15, a spliceosome-associated protein, acts as a positive regulator of miRNA biogenesis.CWC15 binds the promoters of genes encoding miRNAs (MIRs), promotes their activity, and increases the occupancy of DNA-dependent RNA polymerases at MIR promoters, suggesting that CWC15 positively regulates the transcription of primary miRNA transcripts (pri-miRNAs).In addition, CWC15 interacts with Serrate (SE) and HYL1, two key components of microprocessor, and is required for efficient pri-miRNA processing and the HYL1-pri-miRNA interaction.Moreover, CWC15 interacts with the 20 S proteasome and PRP4KA, facilitating SE phosphorylation by PRP4KA, and subsequent non-functional SE degradation by the 20 S proteasome.These data reveal that CWC15 ensures optimal miRNA biogenesis by maintaining proper SE levels and by modulating pri-miRNA levels.Taken together, this study uncovers the role of a conserved splicing-related protein in miRNA biogenesis.microRNAs (miRNAs), ~21 nucleotides in size, are important regulator of gene expression and play crucial roles in various biological processes in many eukaryotes including plants and animals [1][2][3][4][5] .miRNAs are generated from their primary transcripts (pri-miRNAs), which contain one or more miRNA-residing imperfect stem-loops [1][2][3][4][5] .Upon production, miRNAs are loaded into the effector protein called Argonaute (AGO) to direct complementary sequence-dependent post-transcriptional gene silencing [1][2][3][4][5] .
Intriguingly, both HYL1 and SE are subjected to post-translational control to fine-tune miRNA biogenesis 34,35 .The activity and stability of HYL1 is modulated by phosphorylation and dephosphorylation 26,[36][37][38][39][40][41][42] .Dephosphorylated HYL1, but not phosphorylated HYL1, promotes miRNA biogenesis 36 .Moreover, phosphorylated HYL1 appears to be more stable in extended periods of darkness and is reactivated by dephosphorylation, suggesting that phosphorylated HYL1 may serve as a reservoir for dephosphorylated HYL1 40 .SE contains intrinsically disordered regions (IDRs) and is typically assembled into the macromolecular complexes for its functions 43 .The unloaded or excessive SE is degraded by 20 S proteasome alpha subunit G1 (PAG1) to ensure proper function of SE 43 .Interestingly, degradation of SE is triggered by the pre-mRNA processing 4 kinase A (PRP4KA)-mediated phosphorylation, which also reduces SE affinity to HYL1 and negatively affects SE-mediated miRNA production 44 .However, how SE phosphorylation and degradation are regulated is still unclear.
Cwf_Cwc_15 domain-containing protein (CWC15) from yeast and its ortholog AD002 from human were identified as a spliceosomeassociated protein and later were shown as a component of the core machinery of the spliceosome [45][46][47] .AD002 is associated with the conserved hPrp19/CDC5L complex 45 , which is required for catalytic activation of the spliceosome 48 .However, CWC15 was not found in the NineTeen Complex (NTC), the yeast counterpart of PRP19 45 .CWC15/ AD002 is essential for the development of yeast and metazoans 49 .In Arabidopsis, lack of CWC15 also causes embryo lethality, while downregulation of CWC15 results in growth defects accompanied with moderate splicing defects 50 .However, the functional mechanism of CWC15 is still poorly understood.
Here, we show that CWC15 plays important roles in miRNA biogenesis.Knockdown of CWC15 with an artificial miRNA (amiR CWC15 ) reduced the accumulation of miRNAs and pri-miRNAs.CWC15 binds MIR promoters, and promotes Pol II occupancy at MIR promoters, suggesting that CWC15 positively regulates pri-miRNA transcription.In addition, CWC15 interacts with HYL1 and SE, but not DCL1, facilitates HYL1-pri-miRNA interaction and is required for efficient pri-miRNA processing.Intriguingly, SE protein levels are increased in amiR CWC15 , which is caused by decreased 20 S proteasome activity.Consistent with this observation, CWC15 interacts with both 20 S proteasome and with PRP4KA, and promotes SE phosphorylation and degradation.These results suggest that CWC15 may serve as a platform to facilitate degradation of unpacked or excessive SE by promoting its phosphorylation, which ensures proper miRNA biogenesis.Taken together, this study shows that CWC15 plays multifaceted roles in miRNA biogenesis.

CWC15 may function in miRNA biogenesis
To better understand the mechanisms governing miRNA biogenesis, we analyzed the network of CDC5, which is a core component of MAC, and promotes pri-miRNA transcription and processing 28 , using the STRING program (https://string-db.org) with a high confidence score 51 .We expected to identify candidate proteins involved in miRNA biogenesis, based on the assumption that functional related proteins often co-exist in the same network.CDC5 network consists of known MAC components and several proteins involved in RNA metabolisms (Supplementary Fig. 1).Among candidate proteins, we focused on a CWC15 (EMB2769; AT3G13200), (Supplementary Fig. 1), because its potential interaction with the MAC complex 50 .CWC15 is a conserved protein in eukaryotes (Supplementary Fig. 2a) and contains a Cwf_Cwc_15 domain of unknown function (Supplementary Fig. 2b).Protein structure prediction found that the Cwf_Cwc_15 domain contains a disordered region and a coiled-coil motif (Supplementary Fig. 2b).
To evaluate its function, we first examined the association of CWC15 with MAC3A, CDC5, MAC5A using a bimolecular fluorescence complementation (BiFC) assay.In this assay, we transiently coexpressed CWC15 fused with the N-terminal fragment of yellow fluorescent protein (nYFP) with PRL1, CDC5, MAC3A or MAC5A fused with the C-terminal fragment of YFP (cYFP), in the leaves of Nicotiana benthamiana (N.benthamiana).Co-expression of nYFP-CWC15 with cYFP tagged MAC components resulted in YFP signals (Supplementary Fig. 2c), suggesting the possible association of CWC15 with the MAC complex.Interestingly, MAC5 formed speckles with CWC15 while CDC5 and MAC3A did not.A possible explanation is that MAC5, but not MAC3A and CDC5, contains unstructured region, which is known to promote speckles formation through liquid-liquid phase separation 52 .Moreover, CWC15 also co-IPed with MAC5A (Supplementary Fig. 2d).Taken together, these results confirm that CWC15 is correlated with MAC and may function in miRNA biogenesis.

CWC15 is required for miRNA accumulation
Next we tested if CWC15 is required for miRNA accumulation.Because lack of CWC15 causes embryo lethality 50 , we used an artificial miRNA (amiR CWC15 ) targeting 5' UTR (Untranslated region) to knockdown the transcript levels of CWC15 in Col (wild-type; WT) (Fig. 1a).In T1 generation, we identified amiR CWC15 −1, −2 and −3 lines, in which the transcript levels of CWC15 were reduced (Supplementary Fig. 3a).These lines displayed pleiotropic development defects such as smaller plant size and serrated leaves (Fig. 1b and Supplementary Fig. 3b).Expression of a CWC15 open reading frame fused with a GFP tag at its C-terminal driven by its native promoter (pCWC15::GFP-CWC15) in amiR CWC15 −1 line fully rescued the developmental defects of amiR CWC15 (Supplementary Fig. 3b), demonstrating that the growth defects of amiR CWC15 are caused by reduced CWC15 transcript levels.
We next examined the effect of amiR CWC15 on miRNA accumulation by Northern blot.All examined miRNAs were reduced in abundance in two amiR CWC15 lines relative to Col (Fig. 1c).In addition, stem-loop RT-qPCR 53 analysis showed that the reduced miRNA levels were fully recovered in the complementation lines (Fig. 1d), suggesting that CWC15 is required for miRNA accumulation.Next, we analyzed the effect of CWC15 on miRNA accumulation at global levels by comparing miRNA profile in amiR CWC15 with that in Col through Illumina deep sequencing analyses.The result showed that many miRNAs displayed reduced accumulation in amiR CWC15 line (Fig. 1e and Supplementary dataset 1) with a median log2 fold change −0.5 relative to Col. Taken together, these results demonstrate that CWC15 is required for miRNA accumulation.
We further performed reverse transcription quantitative PCR (RT-qPCR) to examine the transcript levels of several miRNA target transcripts including CMT3, ARF8, APS3, CKB3, PHV and SPL3/9, which are targets of miR823, miR167, miR395, miR397, miR166 and miR156, respectively.The transcript levels of these targets were moderately increased in amiR CWC15 relative to Col and recovered in the complementation lines (Fig. 1f), agreeing with the decreased levels of miRNAs in amiR CWC15 .

CWC15 promotes pri-miRNA transcription
Next, we asked how CWC15 promotes miRNA accumulation.We first examined the impact of CWC15 on pri-miRNA accumulation, which is one of the factors determining miRNA levels.RT-qPCR analysis showed that all examined pri-miRNAs were reduced in abundance in amiR CWC15 relative to Col (Fig. 2a and Supplementary Fig. 4).Moreover, the pri-miRNA levels were fully recovered in the complementation lines (Supplementary Fig. 4a), suggesting that CWC15 enhances pri-miRNA accumulation.Because pri-miRNA levels are partially determined by transcription, we asked if CWC15 could affect MIR promoter activity using a transgenic line harboring a GUS gene driven by the MIR167a promoter (pMIR167a::GUS), which has been used as a reporter for MIR transcription 23 .We crossed pMIR167a::GUS into amiR CWC15 and compared GUS expression levels in amiR CWC15 with those in WT.GUS staining and RT-qPCR showed that GUS levels were reduced in amiR CWC15 (Fig. 2b, c), suggesting that CWC15 may modulate MIR promoter activity.To validate the result, we next test the effect of CWC15 on the occupancy of Pol II at MIR promoters using chromatin immunoprecipitation (ChIP) assay with anti-RPB2 (the second largest subunit of Pol II) antibodies.The results showed that amiR CWC15 reduced the occupancy of Pol II at MIR promoters (Fig. 2d).To test if CWC15 directly affects MIR transcription, we examined the interaction of CWC15 with RBP2 using co-IP.However, RBP2 did not co-IPed with CWC15 (Fig. 2e).We next examined the occupancy of CWC15 at MIR promoters by ChIP analysis of the transgenic plants harboring a p35S::2HA-CWC15 transgene with antibodies recognizing HA epitope tag.qPCR detected the enrichment of examined MIR promoter fragments in HA-CWC15 IPs (Fig. 2f).Taken together, these results show that CWC15 plays a role in regulating MIR transcription.
Because CWC15 is a splicing factor, we also examined its effect of CWC15 on pri-miRNA splicing.RT-PCR analysis showed that the splicing of some pri-miRNAs was impaired in amiR CWC15 (Supplementary Fig. 4b).

CWC15 promotes the cleavage activity of the DCL1 complex
The interaction of CWC15 with HYL1 and SE suggested that CWC15 might have a role in modulating DCL1 activity.We thus examined if CWC15 could modulate pri-miRNA cleavage by the DCL1 complex using an in vitro assay 54 .As previously described, [ 32 P] labeled pri-miR162b (MIR162b) harboring the stem-loop of miR162b with 6-nt arms at each end and pre-miR162b (Fig. 4a, b) were incubated with protein extracts from young flowers of amiR CWC15 or Col to evaluate the production of miR162b (supplementary Fig. 5).The result showed that the amount of miR162b processed from radioactive labeled MIR162b or pre-miR162b was lower in amiR CWC15 than in Col (Fig. 4c, d and supplementary Fig. 5).At 120 min time point, miR162b levels produced from MIR162b and pre-miR162b in amiR CWC15 were ~20% and ~45% of those generated in Col, respectively (Fig. 4e, f), suggesting that CWC15 may promote the optimal activity of the DCL1 complex.

CWC15 promotes the interaction of HYL1 with pri-miRNAs
The less impact of CWC15 on pre-miR162b than on MIR162b suggests that CWC15 may have additional roles in pri-miRNA processing.One possibility is that CWC15 might help the interaction of pri-miRNAs with the DCL1 complex given its interaction with SE and HYL1.We compared the association of pri-miRNAs with HYL1 in amiR CWC15 with that in Col, which has been considered as indicator for the loading of pri-miRNA to the DCL1 complex, by RNA immunoprecipitation (RIP) using antibodies recognizing HYL1.Similar amount of HYL1 was IPed from amiR CWC15 and Col (Fig. 4g).RT-qPCR revealed that the amounts of examined pri-miRNAs (MIR172b, MIR167a, MIR159a, MIR173, MIR156b and MIR156a) in HYL1 IPs from amiR CWC15 were lower than those from Col (Fig. 4h).

CWC15 promotes the degradation of SE proteins
To understand how CWC15 affects DCL1 activity, we examined the effect of CWC15 on the transcript levels of DCL1, DDL, SE, HYL1, CBP20/80 and HEN1, which function in miRNA biogenesis, through RT-qPCR.The transcript levels of these genes slightly increased in amiR CWC15 (Supplementary Fig. 6).We further evaluated the proteins levels of DCL1, SE and HYL1 and found that SE and DCL1 protein levels were increased in amiR CWC15 relative to those in Col, while HYL1 protein levels were not altered (Fig. 5a).Because SE is also subjected to degradation, we examined if the increased SE protein levels in amiR CWC15 were caused by increased stability 43 .Indeed, after treatment with MG132, which is a proteasome inhibitor known to block SE degradation, the abundance of SE in Col was comparable to that in amiR CWC15 (Fig. 5b), suggesting that the decay of SE in amiR CWC15 may be disrupted.To validate this result, we treated cell lysates from Col and amiR CWC15 seedlings with cycloheximide (CHX) to block protein synthesis and examined the half-life of SE.Agreeing with previous report, the half-life of SE in Col was ~10 min (Fig. 5c).In contrast, SE half-life in amiR CWC15 was extended to ~20 min (Fig. 5c), suggesting that CWC15 promotes the degradation of SE.

CWC15 is required for the 20 S proteasome-mediated SE degradation
We next asked if the increased SE stability is caused by impaired 20 S proteasome activity, which degrades SE 43 .We used an in vitro 20 S proteasome assay, in which the 20 S proteasome was isolated through immunoprecipitation of the PAG1 complex 43 , to test this possibility.Col plants harboring a PAG1-FlAG-4MYC transgene driven by its native promoter (pPAG1::PAG1-FM) was crossed with amiR CWC15 to obtain amiR CWC15 containing pPAG1::PAG1-FM.The protein levels of PAG1 in amiR CWC15 were like those in Col (Supplementary Fig. 7a, b).We IPed the PAG1 complex (Fig. 5d).Silver stain assay showed that 20 S proteasomes were purified successfully (Fig. 5e).Moreover, the purified 20 S proteasomes was able to degrade recombinant SE protein in vitro and its activity was inhibited by MG132 (Supplementary Fig. 7c, d), agreeing with previous report 43 .Thus, we used this system to test the effect of amiR CWC15 on the 20 S proteasome-mediated SE degradation.The result showed that the degradation rate of SE by the 20 S proteasome from amiR CWC15 was lower than that from Col (Fig. 5d-f), demonstrating that CWC15 is required for the 20 S proteasome-mediated SE degradation.Because impaired 20 S proteasome activity leads to the accumulation of SE in cytoplasm 43 , we asked if amiR CWC15 had a similar effect using a nuclear-cytoplasmic fractionation assay.The result showed that the ratio of cytoplasm-localized SE in amiR CWC15 was increased relative to Col, in which SE was mostly localized in the nucleus (Fig. 5g, h)

CWC15 associates with the 20 S proteasome to promote SE degradation
The effect of CWC15 on SE degradation promoted us to test if CWC15 is associated with the 20 S proteasome using the BiFC assay.Among four selected 20 S proteasome subunits known to interact with SE 43 , CWC15 interacted with PAB1, PAG1 and PBA1, but not PBE1, in the nucleus (Fig. 6a).It should be noted that CWC15-PAG1 BiFC signal could also be observed in the cytoplasm.Agreeing with this result cell fractionation assay showed that CWC15 localized at both cytoplasm and nucleus (Supplementary Fig. 8).
The association of CWC15 with SE and the 20 S proteasome raised at least two possible ways by which CWC15 affects SE degradation.It may facilitate the recruitment of SE to the 20 S proteasome and/or promotes the 20 S proteasome activity on SE.In first scenario, we would expect a reduction of SE amount in the 20 S proteasome of amiR CWC15 relative to Col, while in the second scenario, SE will be accumulated in the 20 S proteasome of amiR CWC15 .To test these two possibilities, we IPed PAG1-FM from transgenic lines expressing PAG1-FM (Fig. 6d).Similar amount of PAG1 was IPed from Col and amiR CWC15 (Fig. 6d).The amount of SE in the PAG1 complex from amiR CWC15 were ~2.1 times that from Col (Fig. 6d).Moreover, the amount difference of SE in the PAG1 complex between Col and amiR CWC15 was reduced after treatment with MG132 (Fig. 6d).These data suggest that CWC15 may promote 20 S proteasome activity on SE through its association with the 20 S proteasome and SE.

CWC15 associates with PRP4KA to promote SE phosphorylation
Because SE degradation is triggered by PRP4KA-mediated phosphorylation, we sought to test if CWC15 promotes SE degradation by modulating phosphorylation.To examine this possibility, we first tested the interaction of CWC15 with PRP4KA using BIFC.Co-expression of cYFP-CWC15/ nYFP-PRP4KA or nYFP-CWC15/ cYFP-PRP4KA resulted in YFP signal (Fig. 7a).Moreover, CWC15 and PRP4KA reciprocally pulled down each other when co-expressed (Fig. 7b, c), showing the CWC15-PRP4KA interaction.This result promoted us to test if CWC15 affected SE phosphorylation.SE phosphorylation status was analyzed using phos-tag gels, which contain a phos-tag that binds phosphatecontaining molecules resulting in reduced mobility of phosphorylated proteins.The results showed that the phosphorylated form of SE was reduced in amiR CWC15 relative to Col plants (Fig. 7d, e).Taken together, these results show that CWC15 interacts with PRP4KA and promotes its activity on SE.

Discussion
CWC15 is a conserved protein in eukaryotes spliceosome [45][46][47] .In yeast, CWC15 interacts with several U5 snRNA components and was suggested to stabilize the spliceosomal core [45][46][47] .Lack of CWC15 causes lethality in yeast, Cattle, and Arabidopsis, suggesting its essential role in development.However, the exact function of CWC15 in development and RNA metabolism including splicing remains to be identified.In this study, we find that knockdown of CWC15 results in reduced miRNA accumulation.In addition, CWC15 interacts with SE and HYL1, and binds MIR loci.These results support that CWC15 directly acts in miRNA biogenesis, which shall partially contribute to its essential role in development.
SE is an IDP containing IDRs, which act as scaffolds for formation of D-bodies 17 , and needs to be assembled into complexes for its normal function 43 .Homeostasis of SE levels is crucial for its proper function 43 .In Arabidopsis, it has been shown that excess amount of or unpacked SE disrupts the formation of the D-bodies, impairing miRNA biogenesis 43 .We find that the rate of SE degradation is slower in amiR CWC15 relative to Col.Moreover, the accumulation of SE is increased in the cytoplasmic fraction in amiR CWC15 , which resembles the observation in pag1 43 , suggesting that CWC15 is required for the degradation of nonfunctional SE (Fig. 7f).In Arabidopsis, the 20 S proteasome is responsible for the degradation of SE.The reduced degradation rate of SE in amiR CWC15 promoted us to examine the interaction between CWC15 and the 20 S proteasome.Indeed, CWC15 is associated with the 20 S proteasome.The interaction of CWC15 with both 20 S proteasome and SE raises the possibility that CWC15 may deliver SE to 20 S proteasome.However, this is unlikely the case because the accumulation of SE in the inactivated 20 S proteasome from amiR CWC15 is not reduced compared with Col. Thus, CWC15 may promote the activity of 20 S proteasome on SE (Fig. 7f).Efficient SE degradation requires phosphorylation by PRP4KA.We find that the ratio of nonphosphorylated to phosphorylated SE is significantly lower in amiR CWC15 .In addition, CWC15 interacts with PRP4KA.These results suggest that CWC15 facilitates SE degradation by promoting its phosphorylation (Fig. 7f).In addition, CWC15 may promote SE degradation by its interaction with the 20 S proteasome.
Previous study showed that the 20 S proteasome and PRP4KA interact with SE in the nucleus, suggesting that SE is phosphorylated by PRP4KA in the nucleus and subsequently degraded by 20 S proteosome at the nucleus 43,44 .However, SE is accumulated in cytoplasm of pag-1 and prp4ka.It was proposed that the accumulation of SE maybe due to the reprogramming of protein trafficking genes in pag-1 43 .BiFC analysis suggested that CWC15 mainly interacts with the 20 S proteasome and PRP4KA in the nucleus, showing that it may promote their activity in the nucleus.CWC15 also localizes in the cytoplasm, indicating that it may also affect 20 S proteasomemediated reprograming of protein trafficking genes.Alternatively, it may help SE degradation in cytoplasm.It shall be noted that SE transcript levels are increased in amiR CWC15 , which may also contribute to increased SE protein levels.
SE acts as a platform to form the D-bodies and/or to recruit the D-body to pri-miRNAs, or vice versa 17,55 .We suspect that excess amount of SE in amiR CWC15 may affect the formation of active DCL1 complex and/or the recruitment of pri-miRNA to the DCL1 complex.Indeed, pri-miRNA processing efficiency and HYL1-pri-miRNA interaction are reduced in amiR CWC15 .CWC15 may also have additional roles in modulating pri-miRNA processing.In yeast, CWC15 stabilizes the interaction between U6 RNA and PRP8 56 .By analogy, CWC15 may be able to stabilize HYL1-pri-miRNA interaction.In addition, CWC15 contains a  coiled-coil domain, suggesting that it may bind pri-miRNAs affecting miRNA biogenesis.
Pri-miRNAs are co-transcriptionally processed.The processing and transcription are coordinated by several protein factors including NOT2 (Negative on TATA less2) 57 , Elongator 58 , PRL1/2 27,31 , CDC5 29 , MAC3 29,31 , MAC7 31 and SNC1 (SUPPRESSOR OF npr1-1, CONSTITUTIVE 1) 59 , which interact with pol II and the DCL1 complex.CWC15 binds MIR promoter, promotes MIR promoter activity, is required for Pol II occupancy at MIR loci and interacts with the DCL1 complex strongly support that CWC15 positively regulate MIR transcription and links pri-miRNA transcription with processing (Fig. 7f).We envision that CWC15 may facilitate pri-miRNA transcription at MIR locus, where it removes unpacked/unfolded SE to help HYL1-pri-miRNA interaction and/or to promote efficient pri-miRNA processing (Fig. 7f).CWC15 does not interact with RBP2, suggesting that CWC15 may indirectly recruit Pol II.One possibility is that CWC15 may modulate pri-miRNA transcription through its interaction with CDC5 (Fig. 7f), which directly interacts with Pol II and recruits Pol II to MIR locus 28 .
We noticed that some miRNAs are upregulated in amiR CWC15 .A possible explanation is that these miRNAs may be derived from pri-miRNAs harboring specific features.However, sequence analysis did not identify such feature.Alternatively, it could be due to the function of CWC15 in splicing, which may control the biological processes that indirectly affect the expression of specific miRNAs.Supporting this notion, the accumulation of some miRNAs also is increased in other mac mutants [27][28][29]31 . Intrestingly, CWC5 differentially affects the accumulation of miRNAs produced from two arms of some pri-miRNAs (Supplementary dataset 1).For instance, the abundance of miR-171c-3p, miR-167a-5p and miR-167c-5p was decreased in amiR CWC15 , while the amount of miR-171-5p miR-167a-3p and miR-167c-3p were increased in amiR CWC15 .Studies have revealed that the differential accumulation of two arms from the same precursor is controlled at processing or postprocessing levels 60,61 .Given the association of CWC15 with the DCL1 complex, we suspect that CWC15 could play a role in modulating the arm-specific processing of some pri-miRNAs.
In summary, we uncover an activity of CWC15 outside splicing.It associates with pri-miRNA processing complex and enhances pri-miRNA processing by facilitating the degradation of excess amount of SE and promoting the interaction of HYL1 with pri-miRNAs.In addition, CWC15 positively contributes to pri-miRNA transcription by promoting the occupancy of Pol II at MIR promoters.CWC15 also functions in splicing in Arabidopsis.It may act like its counterparts from human and yeast to stabilize the splicing machinery [45][46][47] .In Arabidopsis, some pri-miRNAs also contain introns, whose splicing may affect processing by DCL1.It is possible that CWC15, like several other splicing factors, may also affect miRNA biogenesis through modulating splicing of some pri-miRNAs.However, the accumulation of miRNAs originated from both intron and intronless pri-miRNAs is reduced in amiR CWC15 , suggesting that the role of CWC15 in promoting miRNA biogenesis is independent of its function in splicing.CWC15 is a conserved protein in eukaryotes.Like in Arabidopsis, CWC15 might also influence the production of miRNAs in other organisms by modulating orthologs of SE, including the mammalian ARS2.Supporting this notion, the null mutations of CWC15 cause embryo lethality of mammals, which agrees with essential of miRNA in controlling development.

Plant materials and growth condition
Transgenic line containing pPAG1::gPAG1-FM (gPAG1-FM) is in the Columbia (Col-0) genetic background 43 .The transgenic line harboring the pMIR167a::GUS transgene 23 was crossed to amiR CWC15 .In the F2 generation, ten Col-0 and amiR CWC15 plants containing the GUS transgene were selected by PCR-based genotyping and pooled for GUS transcript level analyses.All plants were grown at 22 °C with 16-h light and 8-h dark cycles.

Phylogenetic analysis
For sequence alignment, sequences of interest in the FASTA format were input into the ClustalX 1.8 program and aligned using the ClustalX algorithm 63 .The aligned sequences were then used for phylogenetic analysis using the MEGA6 program 64 .To build an unrooted phylogenetic tree using MEGA6, the evolutionary history was inferred using the neighbor-joining method.The bootstrap consensus tree inferred from 2000 replicates was taken to represent the evolutionary history of the taxa analyzed.Branches corresponding to partitions reproduced in <50% bootstrap replicates were collapsed.The evolutionary distances were computed using the Jones-Thornton-Taylor matrix-based method 65 , with units representing the number of amino acid substitutions per site.The rate variation among sites was modeled with a gamma distribution (shape parameter = 1).

BiFC assay
Paired cYFP-and nYFP-fusion proteins were transiently co-expressed in in N. benthamiana leaves.After 48 h, YFP and chlorophyll autofluorescence signals were observed by a confocal microscopy (Nikon A1 HD25).

GUS histochemical staining
For GUS staining, tissues from amiR CWC15 or WT plants harboring pMIR167a::GUS were incubated for 5 h in the staining solution at 37 °C.Tissue clearing was performed with 70% ethanol.

Dicer activity assay
In vitro dicer activity assay was performed according to 54 .MIR162b or pre-miR162b RNA was prepared by in vitro transcription under the presence of [α-32 P] UTP.For annealing, α-32 P labeled MIR162b or pre-miR162b was heated at 95°C for 5 min in the 37.5 µl annealing buffer (20 mM Tris-HCL pH 7.5; 0.5 M NaCl; 5 mM EDTA) and then kept at ambient temperature for 1 h.Dicer protein extracts were prepared by incubating of 0.1 g flower tissue powder with 400 µl dicer extraction buffer (20 mM Tris-HCL pH 7.5; 4 mM MgCl 2 ; 5 mM DTT) at 4°C for 1 h.After spining at 13000 x g, 4°C for 15 min, the supernatants were collected and used for Dicer activity assay.14 µl protein extracts (2 mg/ml) were mixed with 2 µl annealed MIR162b or pre-miR162b RNA and 4 µl Dicer reaction buffer (0.5 M NaCl; 5 mM ATP; 1 mM GTP; 6 mM MgCl 2 ; 125 mM creatine phosphate, 150 µg/ml creatine kinase and 6 µl RNAsin Rnase inhibitor), and reacted at ambient temperature for various time.The reaction was stopped by adding 50 µl chloroform for RNA extraction.RNA was resolved on a 16% denaturing PAGE gels.Radioactive signals were quantified with ImageQuant (5.2).

Northern Blot and RT-qPCR Analyses
∼15 μg total RNAs extracted from inflorescences were separated on 16% PAGE (polyacrylamide gel electrophoresis) gel and transferred to nylon membranes. 32P-labeled antisense DNA oligonucleotides were used to detect small RNAs.Radioactive signals were detected with a PhosphorImager and quantified with ImageQuant.
The transcript levels of pri-miRNAs, miRNA targets, genes involved in miRNA biogenesis and GUS mRNAs were determined using RT-qPCR.Reverse transcription was performed on DNA-free total RNAs using M-MLV reverse transcriptase (Promega) with oligo-d(T) primers according to manufacturer's instructions.The resulting cDNAs were then used as templates for qPCR on an iCycler apparatus (Bio-Rad) with the SYBR green kit (Bio-Rad).The primers used for PCR are listed in supplementary Table 1.

RIP analyses
RIP was performed as described 67 .Three biological replicates were performed.A total of ∼2 g seedlings of Col or amiR CWC15 were crosslinked with 1% formaldehyde.After quenching with glycine, nuclei were extracted and lysed in lysis buffer (50 mM Tris-HCl, pH 8.0; 10 mM EDTA; 1% SDS; 0.1% PMSF and 1% protease inhibitor cocktail) by sonication five times with Branson Sonifier 250 of 10 s pulses of 20% power and 50 s pause between pulses.Then, equal amounts of proteins from various samples were diluted and incubated with anti-HYL1 antibodies conjugated to protein G agarose beads or protein A/G agarose beads (for no-Ab controls).After reverse cross-linking in the presence of 20 µg proteinase K (Invitrogen) at 65 °C for 1 h, RNAs were extracted and used as templates for qRT-PCR analyses, using primers listed in Supplementary Table 1.

In vitro cell-free protein decay assay
The in vitro cell-free decay assay was carried out as described 43 .Tenday-old seedlings of Col-0 or amiR CWC15 were ground to a fine powder in liquid nitrogen, mixed with two fold volume of lysis buffer (25 mM Tris-HCl, pH 7.5, 10 mM NaCl, 10 mM MgCl 2 and 10% glycerol) and incubated at 4 °C for 30 min.The total protein extracts of each sample were centrifuged twice at 4 °C for 10 min at 12,000 x g and then were adjusted to equal concentrations with the lysis buffer.The final supernatant was supplemented with 0.5 mM CHX and 5 mM ATP, and the mixtures were then divided into two parts.One aliquot was added with 50 µM MG132 and the other with 2% DMSO as a control.The mixtures were then incubated at 22 °C for various times before western blot analysis.

Nuclear-cytoplasmic fractionation assay
Nuclear-cytoplasmic fractionation was performed as described 43 .Tenday-old seedlings of Col-0 and amiR CWC15 were ground to a fine powder in liquid nitrogen and mixed with two volumes of lysis buffer.After the lysates were filtered through two layers of Miracloth, the flowthroughs were centrifuged at 1,500 x g for 10 min at 4 °C.The supernatant parts were then centrifuged again at 10,000 x g for 10 min at 4 °C and collected as cytoplasmic fraction.The pellet parts were washed four times with nuclear resuspension buffer 1 (NRB1, 20 mM Tris-HCl, pH 7.4, 25% glycerol, 2.5 mM MgCl 2 and 0.2% Triton X-100).After washing, the pellet was resuspended with 500 ml of NRB 2 (20 mM Tris-HCl, pH 7.5, 0.25 M sucrose, 10 mM MgCl 2 , 0.5% Triton X-100, 5 mM β-mercaptoethanol and 1X protease inhibitor, Roche).Then, 500 ml of NRB 3 (20 mM Tris-HCl, pH 7.5, 1.7 M sucrose, 10 mM MgCl 2 , 0.5% Triton X-100, 5 mM βmercaptoethanol and 1Xprotease inhibitor) was carefully added on the top of samples, followed by centrifuging at 16,000 x g for 45 min at 4 °C.The final pellet was resuspended in 400 ml of lysis buffer and collected as nuclear fraction.The quality of fractionation was validated with cytoplasmic (Rubisco) and nuclear (H3) markers.

Phos-tag analysis
SE phospho-isoforms were analyzed using phos-tag SDS-PAGE following the manufacturer's instructions (Wako).Briefly, total proteins were separated on a phos-tag SDS-PAGE gel and then transferred to a PVDF membrane.SE was then detected with the SE antibodies.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
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Fig. 3 |
Fig. 3 | CWC15 associates with the DCL1 complex.a The interactions with CWC15 with DCL1, HYL1, SE and DDL analyzed by BiFC.Paired cYFP-and nYFP-fusion proteins were co-expressed in leaves of N. benthamiana.Green color indicates the BiFC signal detected by a confocal microscopy at 48 h after infiltration.Scale bar = 10 μm.Three independent experiments yielded consistent results.b Co-IP between MYC-CWC15 and HYL1−2HA.HYL1-2HA and GFP-2HA were co-expressed with MYC-CWC15 in leaves of N. benthamiana, respectively.IPs were performed using anti-MYC antibodies.HYL1-2HA, GFP-2HA and MYC-CWC15 were detected by western blot.Two independent experiments yielded consistent results.c Co-IP between MYC-CWC15 and SE-2H.SE-2HA and GFP-2HA were co-expressed with MYC-CWC15 in leaves of N. benthamiana, respectively.IPs were performed using anti-MYC antibodies.SE-2HA, GFP-2HA and MYC-CWC15 were detected by western blot.Two independent experiments yielded consistent results.d Co-IP between CWC15 and SE or HYL1 in pCWC15::GFP-CWC15/ amiR CWC15 transgenic plants.Co-IP was performed using anti-GFP antibodies.GFP-CWC15, SE and HYL1 were detected by western blot.Two independent experiments yielded consistent results.Source data are provided as a Source Data file.

Fig. 4 |
Fig. 4 | CWC15 is required for miRNA maturation.a, b Schematic diagram of the MIR162b (a) and pre-miR162b (b) used in vitro processing assay.c and d miR162b produced from MIR162b and pre-miR162b in proteins extracts from inflorescences of amiR CWC15 or Col.The reactions were stopped at various time points as indicated in the picture.e and f Quantification of miR162b production in amiR CWC15 compared to that in Col. Quantification analysis was performed at 80 min.The radioactive signal of miR162b were normalized to input and compared with that of Col-0.The amount of miR162b produced in Col was set as 1.Error bars indicate SD from two biological replicates (n = 2) and data are presented as mean values +/-SD.***P < 0.001 (P = 2.66793E-05 in a; P = 4.18939E-05 in b; two-tailed unpaired t-test).

g
and h The association of HYL1 with pri-miRNAs in amiR CWC15 relative to Col detected by RNA immunoprecipitation.IP was performed with anti-HYL1 antibodies.HYL1 was detected by Western blot.Pri-miRNAs associated with HYL1 were examined by RT-qPCR (h) and normalized to the input.UBQ5 serves as a negative control.w/o Ab. indicates without antibodies.Error bars indicate SD from two biological replicates (n = 2) and data are presented as mean values +/− SD.Asterisks indicate significantly reduced association with pri-miRNAs compared to Col based on P-values (two-tailed unpaired t-test; P < 0.05): MIR172b, 0.002867; MIR167a, 0.000157; MIR156b, 0.020845; MIR156a, 0.027099; MIR159a, 0.000751; MIR173, 0.030127; UBQ5, 0.898117.Source data are provided as a Source Data file.

Fig. 5 |
Fig. 5 | amiRCWC15 affects the degradation of SE. a The protein levels of DCL1, HYL1, SE detected by western blot in Col and amiR CWC15 .Rubisco stained with Coomassie brilliant blue and HSC70 were used as loading control.DCL1 was detected by anti-DCL1(1: 1000 dilutions, rabbit polyclonal, AS194307, Agrisera).Three independent experiments yielded consistent results.b SE levels in Col and amiR CWC15 seedlings with or without MG132 treatment determined by western blot.Rubisco serve as loading control.Two independent experiments yielded consistent results.c In vitro cell-free SE-decay assay.Total proteins from Col and amiR CWC15 were extracted and incubated with CHX (0.5 mM) and ATP (5 mM) for the indicated times.SE levels were determined by western blot.Rubisco and ACTIN2 serve as loading control.Two independent experiments yielded consistent results.d PAG1 IPed from 10 day-old plants detected by western blot using an anti-FLAG antibody.IPed PAG1-FM was eluted with the FLAG peptide.Two independent experiments yielded consistent results.e Silver-staining of IPed PAG1-FM complex resolved in 10% SDS-PAGE.The arrow and bracket indicate PAG1-FM proteins and subunits of the 20 S core proteasome (CP), respectively.Two independent experiments yielded consistent results.f In vitro reconstitution assays of SE degradation via affinity purified 20 S proteasome.Recombinant SE proteins were incubated with IPed PAG1-FM complex from Col or amiR CWC15 harboring pPAG1::gPAG1-FM.The reaction mixture was stopped at the indicated time intervals.The numbers below the gels indicate the relative mean signals of SE proteins at different time points that were normalized to those of the proteins at time 0, where the value was arbitrarily assigned a value of 1. g Cell-fractionation analysis of SE protein from Col and amiR CWC15 plants.Total extraction (T), nuclear fraction (N) and cytoplasmic fraction (C).Western blot analysis was conducted with an anti-SE or anti-HYL1 antibodies.Rubisco stained with Coomassie brilliant blue and histone 3 detected by anti-H3 antibody were used as controls for the cytoplasmic-and nuclear-specific fractions, respectively.h Quantification of the nuclear-cytoplasmic distribution of SE protein.The nuclear and cytoplasmic fraction ratios (N/C) of SE protein in amiR CWC15 were sequentially normalized to that of Col-0, which was arbitrarily assigned a value of 1. Bar graph representing the relative N/C ratios derived from band intensity.Error bars indicate SD between two biological replicates (n = 2) with two-tailed unpaired t-test, p-values indicated above plot.Source data are provided as a Source Data file.

Fig. 6 |
Fig. 6 | CWC15 promotes 20 S proteasome activity.a The protein interactions analyzed by BiFC.Paired cYFP-and nYFP-fusion proteins were co-expressed in tobacco leaves.Green color indicates the BiFC signal detected by a confocal microscopy at 48 h after infiltration.Scale bar = 10 μm.Three independent experiments yielded consistent results.b The association of CWC15 with PAG1 and PAB1 detected by Co-IP.PAG1−2HA, PAB1-2HA and GFP-2HA were co-expressed with MYC-CWC15 in leaves of N. benthamiana, respectively.IPs were performed using anti-MYC antibodies.PAG1-2HA, PAB1-2HA, GFP-2HA and MYC-CWC15 were detected by western blot.Two independent experiments yielded consistent results.c Co-IP between CWC15 and PAG1 in plants containing the p35S::2HA-CWC15 and pPAG1::gPAG1-FM transgenes.2HA-CWC15 and PAG1-FM were detected by western blot.Numbers on top of the pictures indicate plants used: 1, 2HA-CWC15/gPAG1-FM; 2, Col-0; 3, gPAG1-FM.Two independent experiments yielded consistent results.d The enrichment of SE in the PAG1 complex with or without MG132 treatment.IP was performed using anti-FLAG antibodies.SE and PAG1-FM were detected by western blot using anti-SE and anti-FLAG, respectively.Numbers on top of the pictures indicate plants used: 1, Col-0; 2, gPAG1-FM, 3, amiR CWC15 /gPAG1-FM.The enriched SE protein compared with IPed PAG1-FM in amiR CWC15 was normalized to that of Col-0, which was assigned a value of 1. Two independent experiments yielded consistent results.Source data are provided as a Source Data file.

Fig. 7 |
Fig. 7 | CWC15 promotes phosphorylation of SE. a Interaction between CWC15 and PRP4KA detected by BiFC.Paired cYFP-and nYFP-fusion proteins were coexpressed in leaves of N. benthamiana.Green color indicates the BiFC signal detected by a confocal microscopy at 48 h after infiltration.Scale bar = 10 μm.Three independent experiments yielded consistent results.b, c Co-IP between CWC15 and PRP4KA.2HA-CWC15 was co-expressed with FM-PRP4KA or GFP−2FLAG in N. benthamiana leaves, respectively.IPs were performed using anti-FLAG antibodies or and anti-HA antibodies.2HA-CWC15, FM-PRP4KA and GFP-2FLAG were detected by western blot.Two independent experiments yielded consistent results.d Phosphorylation status of endogenous SE in amiR CWC15 and Col-0.SE was detected by SDS-PAGE and phosphor-tag gel in parallel using anti-SE antibodies.e Phosphorylation levels of SE in amiR CWC15 relative to Col. Phosphorylated SE proteins were compared with total SE proteins (set as 1) in Col and amiR CWC15 , respectively.Error bars indicate SD from two biological replicates (n = 2) and data are presented as mean values +/-SD.Asterisks indicate significantly reduced phosphorylation compared to Col-0 plants based on P-value (two-tailed unpaired ttest; P < 0.01): 0.002787.f The proposed action model of CWC15 in miRNA biogenesis.CWC15 binds the MIR promoters to facilitate the recruitment of Pol II, which may depend on the function of CDC5.CWC15 interacts with SE and HYL1 to enhance pri-miRNA processing.Moreover, CWC15 interacts with PRP4KA and the 20 S proteosome to promote phosphorylation-dependent SE degradation.Source data are provided as a Source Data file.